The goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. The new TOF-TOF produces high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced are interpreted with corresponding speed. A prototype TOF-TOF instrument for demonstrating feasibility of a high-performance MALDI instrument that provides the speed, sensitivity, and dynamic range required for high-throughput proteomics was designed and constructed in phase I. The essential elements of this new instrument include a new simplified MALDI ion source designed to operate routinely 24/7 with 5 khz laser;a high-resolution first stage MS that provides mono-isotopic precursor selection up to at least m/z 4000 with no measurable loss in ion transmission and less than 1% transmission of adjacent masses;a new TOF-TOF analyzer incorporating high-resolution precursor selection and multiplex operation that allows multiple precursors to be selected and analyzed following each laser shot;and a high resolution second stage MS that incorporates automatic internal calibration of fragment spectra providing high mass accuracy on all fragment masses. Design and performance of these elements are described in the progress report for phase I. The goal of phase II of this project is to complete the development of a fully functional, commercially viable MS-MS system including all of these elements and to demonstrate the performance in applications to biological samples. The remaining work requires not only finalizing the design and construction of the complete system, including electronics and automatic controls, but also demonstrating the long-term reliability and ruggedness of the resulting elements and system. Final testing of the prototypes developed in phase II will be carried out in laboratories of our collaborators on applications requiring rapid and reliable analysis of biological samples with high sensitivity and broad dynamic range. In phase III a high-throughput, high-resolution, multiplexed MALDI TOF-TOF mass spectrometer with the attributes described above will be commercialized, rendering all previous MALDI MS-MS instruments obsolete. Addition of separations interfaces and automation developed in related projects provide integrated systems that may replace electrospray LC-MS-MS systems for many applications. PUBLIC HEALTH RELEVANCE: It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required and no current MS-MS technique comes within an order of magnitude of the required speed and sensitivity.